US20230392119A1 - Compositions and methods for preparing engineered lymphocytes for cell therapy - Google Patents

Compositions and methods for preparing engineered lymphocytes for cell therapy Download PDF

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US20230392119A1
US20230392119A1 US18/324,012 US202318324012A US2023392119A1 US 20230392119 A1 US20230392119 A1 US 20230392119A1 US 202318324012 A US202318324012 A US 202318324012A US 2023392119 A1 US2023392119 A1 US 2023392119A1
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cells
canceled
cell
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car
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Waleed HASO
Qi Cai
Thanh Nguyen Yip
Hsing-Chuan Tsai
Carmen Warren
Melody Geragosian
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Kite Pharma Inc
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Kite Pharma Inc
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Assigned to KITE PHARMA, INC. reassignment KITE PHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NGUYEN YIP, Thanh, GERAGOSIAN, Melody, HASO, Waleed, WARREN, Carmen, CAI, Qi, TSAI, HSING-CHUAN
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464424—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A61K39/464442—Chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present disclosure relates to the field of cell therapy, and more specifically, compositions and methods for manufacturing engineered lymphocytes.
  • Immune cells can be modified to target and kill cancer cells in a patient.
  • methods have been developed to engineer immune cells to express constructs which direct the immune cells to a particular target cancer cell.
  • CARs Chimeric antigen receptors
  • TCRs engineered T cell receptors
  • the current autologous CAR cell manufacturing process typically takes about 7 days and can be much longer.
  • the instant disclosure describes improved processes that can be completed within 6 or even 4 days (or within 5 or even 3 days following an enrichment step).
  • the 5-day process i.e., 5 days following enrichment
  • the 5-day process includes transduction preparation and implementation steps with a higher number of lymphocytes in contact with vectors immobilized to recombinant fibronectin coated to the inner surface of a closed system.
  • Such an improved transduction procedure allows a much abbreviated post-transduction cell expansion step.
  • a method for preparing transduced lymphocytes comprising incubating a sample comprising lymphocytes, obtained from a donor subject, with a polynucleotide vector to transduce the lymphocytes to produce transduced lymphocytes; and culturing the sample comprising the transduced lymphocytes for less than 72 hours before the lymphocytes are harvested to produce a harvested sample.
  • the transduced lymphocytes are cultured for less than 48 hours before being harvested. In some embodiments, the transduced lymphocytes are cultured for less than 36 hours before being harvested.
  • the incubation is carried out in a closed system.
  • the closed system has an inner surface area of at least 1500 cm 2 .
  • the closed system has an inner surface coated with a recombinant human fibronectin, wherein the coating is carried out with a solution that comprises about 1-10 ⁇ g/ml of the recombinant human fibronectin.
  • the inner surface is further in contact with a second solution comprising the polynucleotide vector, wherein the second solution has a volume of about 200 mL.
  • the coating further comprises a drain of the second solution.
  • the sample in the closed system comprises at least 1.5 ⁇ 10 8 lymphocytes. In some embodiments, the sample comprises at least 4 ⁇ 10 8 lymphocytes.
  • the lymphocytes are peripheral blood mononuclear cells (PBMCs) or T cells.
  • the harvested sample comprises CD3+ cells.
  • the harvested sample comprises CD4+ and CD8+ T cells.
  • at least 20% of the harvested CD4+ T cells are na ⁇ ve T cells, and no more than 12% of the harvested CD4+ T cells are effector memory T cells.
  • at least 25% of the harvested CD4+ T cells are na ⁇ ve T cells, and no more than 9% of the harvested CD4+ T cells are effector memory T cells.
  • at least 80% of the harvested CD4+ T cells are CCR7+ cells.
  • at most 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells.
  • At least 10% of the harvested CD8+ T cells are na ⁇ ve T cells, and no more than 30% of the harvested CD8+ T cells are effector memory T cells. In some embodiments, at least 20% of the harvested CD8+ T cells are na ⁇ ve T cells, and no more than 20% of the harvested CD8+ T cells are effector memory T cells. In some embodiments, the na ⁇ ve T cells are characterized as CCR7+ and CD45RA+. In some embodiments, the effector memory T cells are characterized as CCR7 ⁇ , CD45RO+ and CD95+. In some embodiments, at least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, at most 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.
  • the method further comprises acquiring the lymphocytes from the donor subject. In some embodiments, the method further comprises enriching the lymphocytes. In some embodiments, the method further comprises contacting the sample with a lymphocyte stimulating agent to activate the lymphocytes.
  • the activating the sample is prior to incubating the sample with the polynucleotide vector.
  • the sample comprises at least 1 ⁇ 10 9 lymphocytes.
  • the activating the sample is after the sample is incubated with the polynucleotide vector.
  • the lymphocyte stimulating agent comprises an anti-CD3 antibody and/or an anti-CD28 antibody.
  • the method further comprises, following the harvesting, administering the harvested lymphocytes to a subject or freezing the harvested lymphocytes.
  • the subject is the same as the donor subject.
  • a total of to 3,000,000 harvested lymphocytes per kilogram of the subject are administered to the subject.
  • a total of 20,000 to 400,000 harvested lymphocytes per kilogram of the subject are administered to the subject.
  • at least 15% of the harvested lymphocytes are transduced with the vector.
  • the polynucleotide vector is a viral vector.
  • the viral vector is a retroviral vector or a lentiviral vector.
  • the vector encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the CAR comprises an intracellular costimulatory domain.
  • the intracellular costimulatory domain is a signaling region of a protein selected from the group consisting of DAP-CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, CDS, GITR, BAFFR,
  • the CAR or TCR recognizes a tumor antigen.
  • the tumor antigen is CD19.
  • the lymphocyte that comprises the CAR is axicabtagene ciloleucel or brexucabtagene autoleucel.
  • the tumor antigen is CD19 and/or CD20.
  • the tumor antigen is CLL-1.
  • the CAR or TCR recognizes an antigen comprising 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), Androgen receptor, B7H3 (CD276), (32-integrins, BAFF, B-lymphoma cell, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), BST2, C242 antigen,
  • a population of cells prepared from a blood sample of a donor subject comprising CD4+ T cells and CD8+ T cells, wherein: at least 20% of harvested the CD4+ T cells are na ⁇ ve T cells, and no more than 12% of the harvested CD4+ T cells are effector memory T cells; at least 10% of the harvested CD8+ T cells are na ⁇ ve T cells, and no more than 30% of the harvested CD8+ T cells are effector memory T cells; at least 50% of the harvested cells are CD3+ T cells; and at least 25% of all T cells are transduced with a polynucleotide vector encoding a CAR or TCR.
  • a population of cells prepared from a blood sample of a donor subject comprising CD4+ T cells and CD8+ T cells, wherein: at least 80% of the CD4+ T cells are CCR7+ cells; at least 60% of the CD8+ T cells are CCR7+ cells; at most 20% of the CD4+ T cells are a combination of effector memory T cells and effector T cells; and at most 40% of the CD8+ T cells are a combination of effector memory T cells and effector T cells are transduced with a polynucleotide vector encoding a CAR or TCR.
  • At least 25% of the harvested CD4+ T cells are na ⁇ ve T cells, and no more than 9% of the harvested CD4+ T cells are effector memory T cells.
  • at least 20% of the harvested CD8+ T cells are na ⁇ ve T cells, and no more than 20% of the harvested CD8+ T cells are effector memory T cells.
  • the na ⁇ ve T cells are characterized as CCR7+ and CD45RA+.
  • the effector memory T cells are characterized as CCR7 ⁇ , CD45RO+ and CD95+.
  • at least 65% of the harvested cells are CD3+ T cells. In some embodiments, at least 15% of all T cells are transduced with the polynucleotide vector.
  • At least 80% of the harvested CD4+ T cells are CCR7+ cells. In some embodiments, at most 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells. In some embodiments, at least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, at most 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.
  • the polynucleotide vector is a viral vector.
  • the viral vector is a retroviral vector or a lentiviral vector.
  • the CAR comprises an intracellular costimulatory domain.
  • the intracellular costimulatory domain is a signaling region of CD28.
  • the CAR recognizes a tumor antigen.
  • the tumor antigen is CD19.
  • the population of cells that comprise the CAR are axicabtagene ciloleucel or brexucabtagene autoleucel.
  • composition comprising the population of cells as described herein.
  • Yet another embodiment provides a method for administering T cells to a subject, comprising injecting to the subject a harvested sample prepared by the method of the present disclosure, or the pharmaceutical composition.
  • the subject has cancer.
  • the cancer is a B cell malignancy.
  • the cancer is Non-Hodgkin's Lymphomas (NHL), Diffuse Large B Cell Lymphoma (DLBCL), Small lymphocytic lymphoma (SLL/CLL), Mantle cell lymphoma (MCL), Follicular lymphoma (FL), Marginal zone lymphoma (MZL), Extranodal (MALT lymphoma), Nodal (Monocytoid B-cell lymphoma), Splenic, Diffuse large cell lymphoma, B cell chronic lymphocytic leukemia/lymphoma, Burkitt's lymphoma, Lymphoblastic lymphoma, acute myeloid leukemia, or multiple myeloma.
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of” can mean within one or more than one standard deviation per the practice in the art. “About” or “comprising essentially of” can mean a range of up to 10% (i.e., ⁇ 10%).
  • “about” can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value.
  • about 5 mg can include any amount between 4.5 mg and 5.5 mg.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • administering refers to the physical introduction of an agent to a subject, such as a modified T cell disclosed herein, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • allogeneic refers to any material derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic T cell transplantation.
  • antibody includes, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen.
  • antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region comprises one constant domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • human antibodies are approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure.
  • the heavy and light chains are linked or connected to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • Naturally-produced antibodies are also glycosylated, e.g., on the CH2 domain.
  • an “antigen binding molecule,” “antigen binding portion,” “antigen binding fragment,” or “antibody fragment” refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived.
  • An antigen binding molecule can include the antigenic complementarity determining regions (CDRs).
  • antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules.
  • Peptibodies i.e., Fc fusion molecules comprising peptide binding domains
  • suitable antigen binding molecule are another example of suitable antigen binding molecule.
  • the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In certain embodiments an antigen binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • the variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • variable region is a human variable region.
  • variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • variable region is a primate (e.g., non-human primate) variable region.
  • variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
  • VL and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof.
  • VH and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.
  • a number of definitions of the CDRs are commonly in use: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering.
  • the AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software.
  • the contact definition is based on an analysis of the available complex crystal structures.
  • autologous refers to any material derived from the same individual to which it is later to be re-introduced.
  • eACTTM engineered autologous cell therapy
  • CAR Chimeric antigen receptor
  • a CAR refers to a molecule engineered to comprise a binding motif and a means of activating immune cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) upon antigen binding.
  • CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors.
  • a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain.
  • a T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell.
  • “Extracellular domain” (or “ECD”) refers to a portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to reside outside of the cell membrane, in the extracellular space.
  • T cell receptor refers to antigen-recognition molecules present on the surface of T cells.
  • TCR antigen-recognition molecules present on the surface of T cells.
  • each of the four TCR genes, ⁇ , ⁇ , ⁇ , and ⁇ may rearrange leading to highly diverse TCR proteins.
  • heterologous means from any source other than naturally occurring sequences.
  • a heterologous sequence included as a part of a costimulatory protein is amino acids that do not naturally occur as, i.e., do not align with, the wild type human costimulatory protein.
  • a heterologous nucleotide sequence refers to a nucleotide sequence other than that of the wild type human costimulatory protein-encoding sequence.
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided polypeptide sequences are known. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences may be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, such as BLAST (basic local alignment search tool).
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).
  • the immune cells of the immunotherapy can come from any source known in the art.
  • immune cells can be differentiated in vitro from a hematopoietic stem cell population, or immune cells can be obtained from a subject.
  • Immune cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • the immune cells can be derived from one or more immune cell lines available in the art.
  • Immune cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation, OPTIPREPTM separation, and/or apheresis. Additional methods of isolating immune cells for an immune cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, which is herein incorporated by reference in its entirety.
  • a “patient” includes any human who is afflicted with a cancer (e.g., a lymphoma or a leukemia).
  • a cancer e.g., a lymphoma or a leukemia.
  • subject and patient are used interchangeably herein.
  • pharmaceutically acceptable refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof.
  • a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient.
  • pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another).
  • a pharmaceutical composition must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient.
  • materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • composition refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.
  • the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population.
  • a pharmaceutical composition may be formulated for administration in solid or liquid form, comprising, without limitation, a form adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or
  • reducing and “decreasing” are used interchangeably herein and indicate any change that is less than the original. “Reducing” and “decreasing” are relative terms, requiring a comparison between pre- and post-measurements. “Reducing” and “decreasing” include complete depletions.
  • reference describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, a reference or control is tested, measured, and/or determined substantially simultaneously with the testing, measuring, or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. When sufficient similarities are present to justify reliance on and/or comparison to a selected reference or control.
  • a “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent, e.g., engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • the vector is a retroviral vector, a DNA vector, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.
  • Treatment or “treating” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.
  • treatment or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission.
  • treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
  • vector refers to a recipient nucleic acid molecule modified to comprise or incorporate a provided nucleic acid sequence.
  • plasmid refers to a circular double stranded DNA molecule into which additional DNA may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors comprise sequences that direct expression of inserted genes to which they are operatively linked.
  • Such vectors may be referred to herein as “expression vectors.” Standard techniques may be used for engineering of vectors, e.g., as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference.
  • the term “7-day process” refers to a CAR cell manufacturing process which takes about 7 days following initial enrichment and/or activation step(s).
  • the 7-day process is at least 8 days in length from the initial enrichment and/or activation step(s) to a harvesting step, and can be between 8 to 11 days in total when including the enrichment and/or activation step(s). In some embodiments, there is no initial activation step, or no activation prior to transduction.
  • the term “5-day process” refers to a CAR cell manufacturing process which takes about 5 days following initial enrichment and/or activation step(s).
  • the 5-day process is 6 days in length from the initial enrichment and/or activation step(s) to a harvesting step, and can be between 6 to 9 days in total when including the enrichment and/or activation step(s). In some embodiments, there is no initial activation step, or no activation prior to transduction.
  • the term “3-day process” refers to a CAR cell manufacturing process which takes about 3 days from initial enrichment and/or activation step(s).
  • the 3-day process is about 4 days in length from the initial enrichment and/or activation step(s) to a harvesting step.
  • the 3-day process does not include a cell expansion step comprising one or more days following a transduction step and preceding a harvesting step. In some embodiments, there is no initial activation step, or no activation prior to transduction.
  • the 3-day process described herein is about 5 days in length from the initial enrichment and/or activation step(s) to a harvesting step. In some embodiments, the 3-day process is about 3 to 4 days in length or about 72 to 96 hours in length from the initial enrichment and/or activation step(s) to a harvesting step (e.g., about 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours in length).
  • the 3-day process is about 4 to 5 days in length or about 96 to about 120 hours in length from the initial enrichment and/or activation step(s) to a harvesting step (e.g., about 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 10 8 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, 120 hours in length). In some embodiments, the 3-day process is less than 5 days or 120 hours in length from the initial enrichment and/or activation step(s) to a harvesting step.
  • the conventional autologous CAR cell manufacturing process takes about 7 days and can be much longer. The lengthy process was believed to be required at least because of the limited supply of starting materials, i.e., lymphocytes obtained from an apheresis collection from a donor subject, the relatively low-efficiency transduction, and the need to expand the transduced cells.
  • starting materials i.e., lymphocytes obtained from an apheresis collection from a donor subject
  • Non-limiting examples of CAR cell manufacturing processes are described in WO2015120096 and WO2016191755, each of which is incorporated herein in its entirety.
  • each washing step during the expansion process considerably reduced the viability of the transduced cells.
  • the several-day expansion process while increasing the total number of transduced cells, reduced the percentages of more juvenile ones (e.g., na ⁇ ve T cells, stem memory T cells, and central memory T cells) and increase the percentages of the more mature ones (e.g., effector memory T cells) (Tables 2 and 6).
  • the more juvenile lymphocytes were shown to be more efficacious than the more mature ones (Tables 5, 10 and 11).
  • the instant disclosure describes improved processes that can be completed within 5 or even 3 days, following an enrichment step.
  • the 5-day process includes transduction preparation and implementation steps with a higher number of lymphocytes in contact with vectors immobilized to recombinant fibronectin coated to the inner surface of a closed system.
  • Such an improved transduction procedure allows a much abbreviated post-transduction cell expansion step.
  • Table 2 transduced cells prepared from the 5-day process skewed towards more juvenile cells. In vivo antitumor efficacy results are shown in (Table 5).
  • the method entails incubating a sample of lymphocytes with a polynucleotide vector to transduce the lymphocytes to produce transduced lymphocytes and culturing the sample that contains the transduced lymphocytes before the lymphocytes are harvested to produce a harvested sample.
  • the culturing step is shortened as compared to the conventional process which takes about 4 days.
  • the culturing step is completed within 96 hours, or within 72 hours, 60 hours, 50 hours, 48 hours, 42 hours, 36 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, or 4 hours.
  • the time for the culturing step is counted from completion of the transduction step (e.g., removal of the cells from the system with immobilized vectors) to harvesting of the cells for storage, transport, or clinical use.
  • Culturing of transduced lymphocytes can be done in media and conditions known in the art.
  • the culturing of the transduced lymphocytes may be performed at a temperature and/or in the presence of CO 2 .
  • the temperature may be about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., or about 39° C.
  • the temperature may be about 34-39° C.
  • the predetermined temperature may be from about 35-37° C.
  • the preferred predetermined temperature may be from about 36-38° C.
  • the predetermined temperature may be about 36-37° C. or more preferably about 37° C.
  • culturing of the transduced lymphocytes may be performed in the presence of a predetermined level of CO 2 .
  • the predetermined level of CO 2 may be 1.0-10% CO 2 .
  • the predetermined level of CO 2 may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO 2 .
  • the predetermined level of CO 2 may be about 4.5-5.5% CO 2 .
  • the predetermined level of CO 2 may be about 5% CO 2 .
  • the predetermined level of CO 2 may be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO 2 .
  • the step of expanding the population of transduced T cells may be performed at a predetermined temperature and/or in the presence of a predetermined level of CO 2 in any combination.
  • the step of expanding the population of transduced T cells may comprise a predetermined temperature of about 36-38° C. and in the presence of a predetermined level of CO 2 of about 4.5-5.5% CO 2 .
  • T cell growth media may be used for culturing the cells in suspension.
  • a T cell growth media may include, but is not limited to, a sterile, low glucose solution that includes a suitable amount of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate.
  • the culturing media is OpTmizerTM (Life Technologies), but one skilled in the art would understand how to generate similar media.
  • the culturing media is EX-VIVOTM serum free media (Lonza Bioscience).
  • the incubation (and/or transduction) step can be carried out in a closed system, without limitation.
  • the closed system is a closed bag culture system, using any suitable cell culture bags (e.g., Mitenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLifeTM Cell Culture bags).
  • the closed system has an inner surface area of at least 500 cm 2 .
  • the closed system has an inner surface area of at least 1000 cm 2 , 1200 cm 2 , 1400 cm 2 , 1500 cm 2 , 1600 cm 2 , 1800 cm 2 , 2000 cm 2 , 2200 cm 2 , 2500 cm 2 , or 3000 cm 2 .
  • the closed system has an inner surface area of not greater than 1500 cm 2 , 1600 cm 2 , 1800 cm 2 , 2000 cm 2 , 2200 cm 2 , 2500 cm 2 , or 3000 cm 2 .
  • the cell culture bags used in the closed system are coated with a recombinant human fibronectin protein.
  • the recombinant human fibronectin fragment may include three functional domains: a central cell-binding domain, heparin-binding domain II, and a CS1-sequence.
  • the recombinant human fibronectin protein or fragment thereof may be used to increase gene efficiency of viral transduction of immune cells by aiding co-localization of target cells or the vector.
  • the recombinant human fibronectin fragment is RetroNectin® (Takara Bio, Japan).
  • the cell culture bags may be coated with recombinant human fibronectin fragment at a concentration of about 0.1-60 ⁇ g/mL, preferably 0.5-40 ⁇ g/mL. In certain embodiments, the cell culture bags may be coated with recombinant human fibronectin fragment at a concentration of about 0.5-20 ⁇ g/mL, 20-40 ⁇ g/mL, or 40-60 ⁇ g/mL.
  • the cell culture bags may be coated with about ⁇ g/mL, 1 ⁇ g/mL, about 2 ⁇ g/mL, about 3 ⁇ g/mL, about 4 ⁇ g/mL, about 5 ⁇ g/mL, about 6 ⁇ g/mL, about 7 ⁇ g/mL, about 8 ⁇ g/mL, about 9 ⁇ g/mL, about 10 ⁇ g/mL, about 11 ⁇ g/mL, about 12 ⁇ g/mL, about 13 ⁇ g/mL, about 14 ⁇ g/mL, about 15 ⁇ g/mL, about 16 ⁇ g/mL, about 17 ⁇ g/mL, about 18 ⁇ g/mL, about 19 ⁇ g/mL, or about 20 ⁇ g/mL recombinant human fibronectin fragment.
  • the cell culture bags may be coated with about 2-5 ⁇ g/mL, about 2-10 ⁇ g/mL, about 2-20 ⁇ g/mL, about 2-25 ⁇ g/mL, about 2-30 ⁇ g/mL, about 2-35 ⁇ g/mL, about 2-40 ⁇ g/mL, about 2-50 ⁇ g/mL, or about 2-60 ⁇ g/mL recombinant human fibronectin fragment.
  • the cell culture bags may be coated with at least about 2 ⁇ g/mL, at least about 5 ⁇ g/mL, at least about 10 ⁇ g/mL, at least about 15 ⁇ g/mL, at least about 20 ⁇ g/mL, at least about 25 ⁇ g/mL, at least about 30 ⁇ g/mL, at least about 40 ⁇ g/mL, at least about 50 ⁇ g/mL, or at least about 60 ⁇ g/mL recombinant human fibronectin fragment.
  • the cell culture bags may be coated with at least about 10 ⁇ g/mL recombinant human fibronectin fragment.
  • the cell culture bags may not be coated with recombinant human fibronectin fragment.
  • a transduction enhancing agent is introduced into the closed system.
  • transduction enhancing agents include VectofusinTM transduction mixtures.
  • the cell culture bags used in the closed bag culture system may be blocked with human albumin serum (HSA). In an alternative embodiment, the cell culture bags are not blocked with HSA.
  • HSA human albumin serum
  • a solution that includes the vector is added to the closed system so that the vector can be immobilized by the recombinant fibronectin, on the inner surface of the closed system.
  • immobilization can improve the transduction efficiency once the cells are added.
  • the vectors can be viral vectors, such as lentiviral vectors, as well as retroviral vectors.
  • viral vectors such as lentiviral vectors, as well as retroviral vectors.
  • Several recombinant viruses have been used as viral vectors to deliver genetic material to a cell.
  • Viral vectors that may be used in accordance with the transduction step may be any ecotropic or amphotropic viral vector including, but not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors.
  • the viral vector is an MSGV1 gamma retroviral vector.
  • the vectors are non-viral vectors.
  • a total volume of at least 100 mL of the solution that contains the vector is used. In some embodiments, a total volume of at least 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, 350 mL, or 400 mL of the solution that contains the vector is used.
  • the vector solution includes at between 1 ⁇ 10 3 to 1 ⁇ 10 12 transduction units per milliliter (TU/ml) of the viral vector.
  • the vector solution can be removed.
  • the closed system does not include recombinant fibronectin.
  • the removal of the vector solution is done by gravity or syringe drain, which helps to retain the immobilized vector on the inner surface while removing impurities.
  • Lymphocyte transduction can be carried in the coated closed system with the immobilized vectors.
  • the transduction is performed with a sample that contained the lymphocytes.
  • the sample includes at least 2.5 ⁇ 10 7 lymphocytes (e.g., T cells).
  • the sample includes at least 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 1.2 ⁇ 10 8 , 1.5 ⁇ 10 8 , 1.8 ⁇ 10 8 , 2 ⁇ 10 8 , 2.2 ⁇ 10 8 , 2.5 ⁇ 10 8 , 2.6 ⁇ 10 8 , 2.7 ⁇ 10 8 , 2.8 ⁇ 10 8 , 2.9 ⁇ 10 8 , 3 ⁇ 10 8 , 3.1 ⁇ 10 8 , 3.2 ⁇ 10 8 , 3.3 ⁇ 10 8 , 3.4 ⁇ 10 8 , 3.5 ⁇ 10 8 , 3.6 ⁇ 10 8 , 3.7 ⁇ 10 8 , 3.8 ⁇ 10 8 , 3.9 ⁇ 10 8 , 4 ⁇ 10 8 , 4.1 ⁇ 10 8 , 4.2 ⁇ 10 8 , 4.3 ⁇ 10 8 , 4.4 ⁇ 10 8 , 4.5 ⁇ 10 8 , 4.6 ⁇ 10 8 , 4.7 ⁇ 10 8 , 4.8 ⁇ 10 8 ,
  • the sample includes no more than 3 ⁇ 10 8 , 3.1 ⁇ 10 8 , 3.2 ⁇ 10 8 , 3.3 ⁇ 10 8 , 3.4 ⁇ 10 8 , 3.5 ⁇ 10 8 , 3.6 ⁇ 10 8 , 3.7 ⁇ 10 8 , 3.8 ⁇ 10 8 , 3.9 ⁇ 10 8 , 4 ⁇ 10 8 , 4.1 ⁇ 10 8 , 4.2 ⁇ 10 8 , 4.3 ⁇ 10 8 , 4.4 ⁇ 10 8 , 4.5 ⁇ 10 8 , 4.6 ⁇ 10 8 , 4.7 ⁇ 10 8 , 4.8 ⁇ 10 8 , 4.9 ⁇ 10 8 , 5 ⁇ 10 8 , 5.1 ⁇ 10 8 , 5.2 ⁇ 10 8 , 5.3 ⁇ 10 8 , 5.4 ⁇ 10 8 , 5.5 ⁇ 10 8 , 5.6 ⁇ 10 8 , 5.7 ⁇ 10 8 , 5.8 ⁇ 10 8 , 5.9 ⁇ 10 8 , 6 ⁇ 10 8 , 6.1 ⁇ 10 8 , 6.2 ⁇ 10 8 , 6.3 ⁇ 10 8 , 6.4 ⁇ 10 8 , 6.5 ⁇
  • lymphocyte transduction is carried out in a closed system that does not contain an immobilizing agent (e.g., recombinant fibronectin).
  • an immobilizing agent e.g., recombinant fibronectin
  • the lymphocytes used in the presently disclosed methods are typically obtained from a donor subject, which may be a cancer patient that is to be treated with a population of cells generated by the methods described herein (i.e., an autologous donor), or may be an individual that donates a lymphocyte sample that, upon generation of the population of cells generated by the methods described herein, will be used to treat a different individual or cancer patient (i.e., an allogeneic donor).
  • the lymphocytes may be obtained from the donor subject by any suitable method used in the art.
  • the lymphocytes may be obtained by any suitable extracorporeal method, venipuncture, or other blood collection method by which a sample of blood and/or lymphocytes is obtained.
  • the lymphocytes are obtained by apheresis.
  • the method described herein further includes a step of enriching a population of lymphocytes obtained from the donor subject, prior to the transduction.
  • Enrichment of lymphocytes may be accomplished by any suitable separation method including, but not limited to, the use of a separation medium (e.g., Ficoll-PaqueTM, RosetteSepTM HLA Total Lymphocyte enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No.
  • a separation medium e.g., Ficoll-PaqueTM, RosetteSepTM HLA Total Lymphocyte enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No.
  • 0850494X a non-ionic iodixanol-based medium such as OptiPrepTM, or the like
  • cell size, shape or density separation by filtration or elutriation immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence activated cell sorting system, FACS), or bead based column separation.
  • immunomagnetic separation e.g., magnetic-activated cell sorting system, MACS
  • fluorescent separation e.g., fluorescence activated cell sorting system, FACS
  • bead based column separation e.g., fluorescence activated cell sorting system
  • circulating lymphoma cells are removed from the sample through positive enrichment for CD4 + /CD8 + cells via the use of selection reagents.
  • incubated cells including cells in which the selection reagent has bound are transferred into a system for immunoaffinity-based separation of the cells.
  • the system for immunoaffinity-based separation is or contains a magnetic separation column.
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation.
  • the isolation in some embodiments includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps may be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use.
  • negative selection may be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step may deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types may simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
  • CD3+, CD28+ T cells may be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • the population of cells is enriched for T cells with na ⁇ ve phenotype (CD45RA+CCR7+).
  • isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection.
  • positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
  • a biological sample e.g., a sample of PBMCs or other white blood cells
  • CD4+ T cells are subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained.
  • CD8+ T cells are selected from the negative fraction.
  • a biological sample is subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained.
  • CD4+ T cells are selected from the negative fraction.
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14.
  • a CD4+ or CD8+selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells.
  • Such CD4+ and CD8+ populations may be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques.
  • the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as DynabeadsTM or MACS beads).
  • the magnetically responsive material, e.g., particle generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
  • a binding partner e.g., an antibody
  • the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.
  • a specific binding member such as an antibody or other binding partner.
  • the incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers.
  • the cells are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added.
  • cell-type specific secondary antibody- or other binding partner e.g., streptavidin
  • streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
  • the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some embodiments, the particles are left attached to the cells for administration to a patient.
  • the magnetizable or magnetically responsive particles are removed from the cells.
  • magnetizable particles are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers.
  • the magnetizable particles are biodegradable.
  • the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto.
  • MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they may be eluted and recovered.
  • the non-target cells are labelled and depleted from the heterogeneous population of cells.
  • the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods.
  • the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination.
  • the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1, which are each incorporated herein by reference.
  • the system or apparatus carries out one or more, e.g., of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion.
  • the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various embodiments of the processing, isolation, engineering, and formulation steps.
  • the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system.
  • Components may include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves.
  • the integrated computer in some embodiments controls components of the instrument and directs the system to perform repeated procedures in a standardized sequence.
  • the magnetic separation unit in some embodiments includes a movable permanent magnet and a holder for the selection column.
  • the peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
  • the CliniMACS system in some embodiments uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution.
  • the cells after labelling of cells with magnetic particles the cells are washed to remove excess particles.
  • a cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag.
  • the tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps.
  • the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
  • separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec).
  • the CliniMACS Prodigy system in some embodiments is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation.
  • the CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers.
  • the CliniMACS Prodigy system may also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports may allow for the sterile removal and replenishment of media and cells may be monitored using an integrated microscope.
  • a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream.
  • a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting.
  • a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells may be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
  • MEMS microelectromechanical systems
  • the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection.
  • separation may be based on binding to fluorescently labeled antibodies.
  • separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system.
  • FACS fluorescence-activated cell sorting
  • MEMS microelectromechanical systems
  • At least 0.5 ⁇ 10 9 lymphocytes are acquired from the donor, and are optionally enriched and/or subjected to the stimulation.
  • at least 0.6 ⁇ 10 9, 0.7 ⁇ 10 9 , 0.8 ⁇ 10 9 , 0.9 ⁇ 10 9 , 1 ⁇ 10 9 , 1.1 ⁇ 10 9 , 1.2 ⁇ 10 9 , 1.3 ⁇ 10 9 , 1.4 ⁇ 10 9 , 1.5 ⁇ 10 9 , 1.6 ⁇ 10 9 , 1.7 ⁇ 10 9 , 1.8 ⁇ 10 9 , 1.9 ⁇ 10 9 , 2 ⁇ 10 9 , 2.5 ⁇ 10 9 , or 3 ⁇ 10 9 lymphocytes are acquired from the donor, and are optionally enriched and/or subjected to the stimulation.
  • no more than 1 ⁇ 10 9 , 1.1 ⁇ 10 9 , 1.2 ⁇ 10 9 , 1.3 ⁇ 10 9 , 1.4 ⁇ 10 9 , 1.5 ⁇ 10 9 , 1.6 ⁇ 10 9 , 1.7 ⁇ 10 9 , 1.8 ⁇ 10 9 , 1.9 ⁇ 10 9 , 2 ⁇ 10 9 , 2.5 ⁇ 10 9 , or 3 ⁇ 10 9 lymphocytes are acquired from the donor, and are optionally enriched and/or subjected to the stimulation.
  • the methods described herein further includes a step of stimulating the lymphocytes with one or more lymphocyte stimulating agents.
  • the stimulation is performed prior to the transduction step.
  • the stimulation is performed after the transduction step.
  • the stimulation step is also referred to herein as an activation step.
  • any combination of one or more suitable lymphocyte stimulating agents may be used to stimulate (activate) the lymphocytes.
  • suitable lymphocyte stimulating agents include an antibody or functional fragment thereof which targets a T-cell stimulatory or co-stimulatory molecule (e.g., anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, or functional fragments thereof) a T cell cytokine (e.g., any isolated, wildtype, or recombinant cytokines such as: interleukin 1 (IL-1), interleukin 2, (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7 (IL-7), interleukin 15 (IL-15), tumor necrosis factor ⁇ (TNF ⁇ )), or any other suitable mitogen (e.g., tetradecanoyl phorbol acetate (TPA), phytohaemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen
  • the lymphocyte stimulating agent may be a bead-based activator, such as T-cell TransActTM (Miltenyi Biotec), Dynabeads® (Thermo Fisher Scientific), or CloudzTM T Cell Activation Kit (R&D Systems).
  • T-cell TransActTM Miltenyi Biotec
  • Dynabeads® Thermo Fisher Scientific
  • CloudzTM T Cell Activation Kit R&D Systems
  • the step of stimulating lymphocytes as described herein may entail stimulating the lymphocytes with one or more stimulating agents at a predetermined temperature, for a predetermined amount of time, and/or in the presence of a predetermined level of CO 2 .
  • the predetermined temperature for stimulation may be about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., or about 39° C.
  • the predetermined temperature for stimulation may be about 34-39° C.
  • the step of stimulating the lymphocytes comprises stimulating the lymphocytes with one or more stimulating agents for a predetermined time. In certain embodiments, the predetermined time for stimulation may be about 24-72 hours.
  • the predetermined time for stimulation may be about 24-36 hours.
  • the step of stimulating the lymphocytes may comprise stimulating the lymphocytes with one or more stimulating agents in the presence of a predetermined level of CO 2 .
  • the predetermined level of CO 2 for stimulation may be about 1.0-10% CO 2 .
  • the predetermined level of CO 2 for stimulation may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO 2 .
  • an anti-CD3 antibody (or functional fragment thereof), an anti-CD28 antibody (or functional fragment thereof), or a combination of anti-CD3 and anti-CD28 antibodies may be used in accordance with the step of stimulating the population of lymphocytes.
  • Any soluble or immobilized anti-CD3 and/or anti-CD28 antibody or functional fragment thereof may be used (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)).
  • the antibodies may be purchased commercially from vendors known in the art including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1 mg/mL, Part No. 170-076-116), and eBioscience, Inc. Further, one skilled in the art would understand how to produce an anti-CD3 and/or anti-CD28 antibody by standard methods. Any antibody used in the methods described herein should be produced under Good Manufacturing Practices (GMP) to conform to relevant agency guidelines for biologic products.
  • GMP Good Manufacturing Practices
  • the T cell stimulating agent may include an anti-CD3 or anti-CD28 antibody at a concentration of from about 20 ng/mL-100 ng/mL.
  • the concentration of anti-CD3 or anti-CD28 antibody may be about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
  • a 3-day process of the present disclosure may comprise the following steps: apheresis material collection, a first wash step conducted on day 0, a second wash step conducted on day 0, an enrichment step conducted on day 0, a third wash step conducted on day 0, an activation step conducted on day 0 up to day 1, a viral transduction step conducted on day 1 up to day 4, and a fourth wash and concentration step conducted on day 3 up to day 4 (the day of harvest).
  • the apheresis material may be fresh apheresis material, cryopreserved apheresis material, or cryopreserved T cells.
  • the 3-day process may comprise an activation step on day 0, day 1, or day 2. In some embodiments, the 3-day process may comprise a viral vector transduction step on day 1, day 2, or day 3.
  • the 3-day process may optionally comprise one, two, three, four, five, six, seven, eight or more wash steps. Each wash step may comprise the same wash procedure or a different wash procedure. In some embodiments, one or more wash steps may occur prior to the enrichment step. In some embodiments, the first wash may occur after the enrichment step. In some embodiments, the first wash may occur after the enrichment step and before the activation step. In some embodiments, the first wash may occur after both the enrichment and activation steps. In some embodiments, a wash step may be conducted on the day of harvest.
  • lymphocytes prepared included higher ratios of juvenile ones (e.g., na ⁇ ve T cells). Accordingly, one embodiment of the present disclosure provides a population of lymphocytes, prepared by the instant methods, that include CD4+ and CD8+ T cells.
  • At least 20% of the CD4+ T cells are na ⁇ ve T cells. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% of the CD4+ T cells are na ⁇ ve T cells.
  • no more than 25% of the CD4+ T cells are effector memory T cells. In some embodiments, no more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or 5% of the CD4+ T cells are effector memory T cells.
  • no more than 44% of the CD4+ T cells are central memory T cells. In some embodiments, no more than 43%, 42%, 41%, or 40% of the CD4+ T cells are central memory T cells.
  • no more than 1.5% of the CD4+ T cells are effector T cells. In some embodiments, no more than 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6% or 0.5% of the CD4+ T cells are effector T cells.
  • At least 80% of the harvested CD4+ T cells are CCR7+ cells. In some embodiments, at most 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells.
  • At least 5% of the CD8+ T cells are na ⁇ ve T cells. In some embodiments, at least 10%, 15%, 20%, 25%, 30% or 35% of the CD8+ T cells are na ⁇ ve T cells.
  • no more than 30% of the CD8+ T cells are effector memory T cells. In some embodiments, no more than 28%, 27%, 25%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10% of the CD8+ T cells are effector memory T cells.
  • no more than 60% of the CD8+ T cells are central memory T cells. In some embodiments, no more than 58%, 56%, 55%, 54%, 52% or 50% of the CD8+ T cells are central memory T cells.
  • At least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, at most 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.
  • na ⁇ ve T cells can be characterized with cell surface markers, as well known in the art.
  • na ⁇ ve T cells can be characterized as CCR7+, CD45RO ⁇ , and CD95 ⁇ . Additional markers for na ⁇ ve T cell include CD45RA+, CD62L+, CD27+, CD28+, CD127+, CD132+, CD25 ⁇ , CD44 ⁇ , and HLA-DR ⁇ .
  • Tscm Surface markers to stem memory T cells include, without limitation, CD45RO ⁇ , CCR7+, CD45RA+, CD62L+(L-selectin), CD27+, CD28+, IL-7Ra+, CD95+, IL-2R ⁇ +, CXCR3+, and LFA ⁇ .
  • Tem Surface markers for effector memory T cells (Tem) include, without limitation, CCR7 ⁇ , CD45RO+ and CD95+. Additional marker for effector memory T cells is IL-2R ⁇ +.
  • suitable markers include CD45RO+, CD95+, IL-2R ⁇ +, CCR7+ and CD62L+.
  • suitable markers include CD45RA+, CD95+, IL-2R ⁇ +, CCR7 ⁇ and CD62L ⁇ , without limitation.
  • venile cells as referred throughout includes one or more of na ⁇ ve T cells, stem memory T cells (Tscm), and central memory T cells (Tcm). These cells are characterized, in part, by expression of CCR7+.
  • the harvested lymphocytes preferably include a good proportion that is CD3+ T cells. In some embodiments, at least 25%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the harvested lymphocytes are CD3+ T cells.
  • the harvested lymphocytes preferably include a good proportion that has been transduced. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 42%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the harvested lymphocytes are transduced with the vector.
  • each transduced lymphocyte includes at least a copy of the vector (or the included coding sequence) integrated to the host genome. In some embodiments, each transduced lymphocyte includes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the vector integrated to the host genome.
  • the vector includes a transgene that encodes a polypeptide.
  • the polypeptide may be a CAR or TCR.
  • the CAR or TCR includes an antigen binding molecule.
  • the antigen binding molecule in some embodiments, has binding specificity to an antigenic moiety.
  • the antigenic moiety is a tumor antigen (e.g., a protein or other molecule that is produced by a cancer cell).
  • the vector includes more than one transgene that encodes for more than one CAR or TCR molecule that include antigen binding molecules with specificity to different antigenic moieties. In some embodiments, the vector includes more than one transgene that encodes for more than one CAR or TCR molecule that include antigen binding molecules with specificity to two different tumor antigens.
  • the antigenic moiety is an antigen associated with a cancer or a cancer cell.
  • antigens may include, but are not limited to, 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin/m, b-catenin/mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster of differentiation 44, exons 7/8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspas
  • tumor antigens include 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), Androgen receptor, B7H3 (CD276), 32-integrins, BAFF, B-lymphoma cell, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), BST2, C242 antigen, 9-0-acetyl-CA19-9
  • the antigenic moiety is associated with virally infected cells (i.e., a viral antigenic moiety).
  • Such antigenic moieties may include, but are not limited to, an Epstein-Barr virus (EBV) antigen (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), a hepatitis A virus antigen (e.g., VP1, VP2, VP3), a hepatitis B virus antigen (e.g., HBsAg, HBcAg, HBeAg), a hepatitis C viral antigen (e.g., envelope glycoproteins E1 and E2), a herpes simplex virus type 1, type 2, or type 8 (HSV1, HSV2, or HSV8) viral antigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL
  • the antigenic moiety is associated with cells having an immune or inflammatory dysfunction.
  • antigenic moieties may include, but are not limited to, myelin basic protein (MBP) myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), pro-insulin, glutamine decarboxylase (GAD65, GAD67), heat shock proteins (HSPs), or any other tissue specific antigen that is involved in or associated with a pathogenic autoimmune process.
  • MBP myelin basic protein
  • PGP myelin proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • CEA carcinoembryonic antigen
  • pro-insulin GAD65, GAD67
  • HSPs heat shock proteins
  • the TCR has specificity to an antigenic moiety on cancer cells.
  • TCR include an anti-707-AP TCR, anti-AFP TCR, anti-ART-4 TCR, anti-BAGE TCR, anti-Bcr-abl TCR, anti-CAMEL TCR, anti-CAP-1 TCR, anti-CASP-8 TCR, anti-CDC27m TCR, anti-CDK4/m TCR, anti-CEA TCR, anti-CT TCR, anti-Cyp-B TCR, anti-DAM TCR, anti-TCR, anti-EGFRvIII TCR, anti-ELF2M TCR, anti-ETV6-AML1 TCR, anti-G250 TCR, GAGE TCR, anti-GnT-V TCR, anti-Gp100 TCR, anti-HAGE TCR, anti-HER-2/neu TCR, anti-HLA-A TCR, anti-HPV TCR, anti-HSP70-2M TCR, anti-HST-2 TCR, anti-
  • a CAR of the present disclosure can include, in addition to the antigen-binding molecule, a hinge, a transmembrane domain, and/or an intracellular domain.
  • the intracellular domain can include a costimulatory domain and an activation domain.
  • a hinge may be an extracellular domain of an antigen binding system positioned between the binding motif and the transmembrane domain.
  • a hinge may also be referred to as an extracellular domain or as a “spacer.”
  • a hinge may contribute to receptor expression, activity, and/or stability.
  • a hinge may also provide flexibility to access the targeted antigen.
  • a hinge domain is positioned between a binding motif and a transmembrane domain.
  • the hinge is, is from, or is derived from (e.g., comprises all or a fragment of) an immunoglobulin-like hinge domain. In some embodiments, a hinge domain is from or derived from an immunoglobulin. In some embodiments, a hinge domain is selected from the hinge of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or a fragment thereof.
  • the hinge is, is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8.alpha., CD8.beta., CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha
  • the hinge is, is from, or is derived from (e.g., comprises all or a fragment of) a hinge of CD8 alpha. In some embodiments, the hinge is, is from, or is derived from a hinge of CD28. In some embodiments, the hinge is, is from, or is derived from a fragment of a hinge of CD8 alpha or a fragment of a hinge of CD28, wherein the fragment is anything less than the whole.
  • a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or C-Terminus, or both, of a CD8 alpha hinge, or of a CD28 hinge.
  • a “transmembrane domain” refers to a domain having an attribute of being present in the membrane when present in a molecule at a cell surface or cell membrane (e.g., spanning a portion or all of a cellular membrane). It is not required that every amino acid in a transmembrane domain be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized in that a designated stretch or portion of a protein is substantially located in the membrane. Amino acid or nucleic acid sequences may be analyzed using a variety of algorithms to predict protein subcellular localization (e.g., transmembrane localization).
  • the programs psort (PSORT.org) and Prosite (prosite.expasy.org) are exemplary of such programs.
  • a transmembrane domain may be derived either from any membrane-bound or transmembrane protein, such as an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49
  • the intracellular domain comprises one or more signaling domains that, upon binding of target antigen to the binding motif, cause and/or mediate an intracellular signal, e.g., that activates one or more immune cell effector functions (e.g., native immune cell effector functions).
  • signaling domains of an intracellular domain mediate activation at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity comprising the secretion of cytokines.
  • signaling domains of an intracellular domain mediate T cell activation, proliferation, survival, and/or other T cell function.
  • An intracellular domain may comprise a signaling domain that is an activating domain.
  • An intracellular domain may comprise a signaling domain that is a costimulatory signaling domain.
  • Intracellular signaling domains that may transduce a signal upon binding of an antigen to an immune cell are known.
  • cytoplasmic sequences of a T cell receptor are known to initiate signal transduction following TCR binding to an antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).
  • suitable signaling domains include, without limitation, those of 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRT AM, cytokine receptor, DAP-DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1,
  • a CAR can also include a costimulatory signaling domain, e.g., to increase signaling potency.
  • a costimulatory signaling domain e.g., to increase signaling potency. See U.S. Pat. Nos. 7,741,465, and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).
  • a signaling domain further comprises one or more additional signaling domains (e.g., costimulatory signaling domains) that activate one or more immune cell effector functions (e.g., a native immune cell effector function described herein).
  • additional signaling domains e.g., costimulatory signaling domains
  • a portion of such costimulatory signaling domains may be used, as long as the portion transduces the effector function signal.
  • a cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or fragment thereof).
  • Non-limiting examples of such T cell co-receptors comprise CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds with CD83.
  • An exemplary costimulatory protein has the amino acid sequence of a costimulatory protein found naturally on T cells, the complete native amino acid sequence of which costimulatory protein is described in NCBI Reference Sequence: NP 0.1.
  • a CAR includes a 4-1BB costimulatory domain.
  • a CAR includes a CD28 costimulatory domain.
  • a CAR includes a DAP-10 costimulatory domain.
  • the costimulatory signaling domain is a signaling domain of CD28.
  • CAR molecules with a CD28 costimulatory signaling domain can particularly benefit from the newly developed, expedited manufacturing process.
  • the CAR is encoded by a nucleic acid molecule embedded in a viral vector.
  • the CAR is encoded by a nucleic acid molecule embedded in a lentiviral vector.
  • the CAR further includes an ITAM.
  • ITAM containing primary cytoplasmic signaling sequences that are of particular use in the disclosure include those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d.
  • the ITAM includes CD3 zeta.
  • the CAR molecule may be any anti-CD19 CAR molecule.
  • the anti-CD19 CAR includes an extracellular scFv domain, an intracellular and/or transmembrane, portion of a CD28 molecule, an optional extracellular portion of the CD28 molecule, and an intracellular CD3zeta domain as described in WO2015120096 or WO2016191755, each of which is incorporated herein in its entirety.
  • the anti-CD19 CAR may also include additional domains, such as a CD8 extracellular and/or transmembrane region, an extracellular immunoglobulin Fc domain (e.g., lgG1, lgG2, lgG3, lgG4), or one or more additional signaling domains, such as 41 BB, OX40, CD2 CD16, CD27, CD30 CD40, PD-1, ICOS, LFA-1, IL-2 Receptor, Fc gamma receptor, or any other costimulatory domains with immunoreceptor tyrosine-based activation motifs.
  • additional domains such as a CD8 extracellular and/or transmembrane region, an extracellular immunoglobulin Fc domain (e.g., lgG1, lgG2, lgG3, lgG4), or one or more additional signaling domains, such as 41 BB, OX40, CD2 CD16, CD27, CD30 CD40, PD-1, ICO
  • the cell surface receptor is an anti-CD19 CAR, such as FMC63-28Z CAR or FMC63-CD828BBZ CAR as set forth in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702, “Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor,” the subject matter of which is hereby incorporated by reference for the purpose of providing the methods of constructing the vectors used to produce T cells expressing the FMC63-28Z CAR or FMC63-CD828BBZ CAR.
  • an anti-CD19 CAR such as FMC63-28Z CAR or FMC63-CD828BBZ CAR as set forth in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702, “Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor,” the subject matter of which is hereby incorporated by reference for the purpose of providing the methods of constructing the vectors used to produce T cells expressing
  • the T cell that includes a CAR molecule is Yescarta® (axicabtagene ciloleucel). In some embodiments, the T cell that includes a CAR molecule is Tecartus® (brexucabtagene autoleucel).
  • the engineered lymphocytes comprise a dual-targeted antigen binding system.
  • Dual-targeted antigen binding systems may comprise bispecific CARs or TCRs and/or bicistronic CARs or TCRs.
  • Bispecific and bicistronic CARs can comprise two binding motifs (in a single CAR molecule or in two CAR molecules, respectively).
  • the vector of the present disclosure encodes bicistronic and/or bispecific CARs (e.g., bicistronic and/or bispecific CARs that bind CD20 and CD19).
  • a pharmaceutical composition that includes a population of engineered lymphocytes produced by the methods described herein.
  • the pharmaceutical composition may also include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting cells of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof.
  • Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • lymphocytes prepared by the instant methods, or the lymphocyte populations as disclosed herein, can be used for treating various diseases and conditions.
  • Such a method may include a step of washing and concentrating the population of engineered lymphocytes with a diluent solution.
  • the diluent solution is normal saline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose/0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof.
  • HSA may be added to the washed and concentrated cells for improved cell viability and cell recovery after thawing.
  • the washing solution is normal saline and washed and concentrated cells are supplemented with HSA (5%).
  • the method may also include a step of generating a cryopreservation mixture, wherein the cryopreservation mixture includes the diluted population of cells in the diluent solution and a suitable cryopreservative solution.
  • the cryopreservative solution may be any suitable cryopreservative solution including, but not limited to, CryoStor10 (BioLife Solution), mixed with the diluent solution of engineered lymphocytes at a ratio of 1:1 or 2:1.
  • HSA may be added to provide a final concentration of about 1.0-10% HSA in the cryopreserved mixture. In certain embodiments, HSA may be added to provide a final concentration of about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% HSA in the cryopreserved mixture. In certain embodiments, HSA may be added to provide a final concentration of about 1-3% HSA, about 1-4% HSA, about 1-5% HSA, about 1-7% HSA, about 2-4% HSA, about 2-5% HSA, about 2-6% HSA, or about 2-7% HSA in the cryopreserved mixture.
  • HSA may be added to provide a final concentration of about 2.5% HSA in the cryopreserved mixture.
  • cryopreservation of a population of engineered T cells may comprise washing cells with 0.9% normal saline, adding HSA at a final concentration of 5% to the washed cells, and diluting the cells 1:1 with CryoStorTM CS10 (for a final concentration of 2.5% HSA in the final cryopreservation mixture).
  • the method also includes a step of freezing the cryopreservation mixture.
  • the cryopreservation mixture is frozen in a controlled rate freezer using a defined freeze cycle at a cell concentration of between about 1e6 to about 1.5e7 cells per mL of cryopreservation mixture.
  • the method may also include a step of storing the cryopreservation mixture in vapor phase liquid nitrogen.
  • Methods and uses are also provided, for treating a disease or pathological condition in a subject having the disease or pathological condition.
  • the method entails administering a therapeutically effective amount or therapeutically effective dose of the engineered lymphocytes to the subject.
  • Pathogenic conditions that may be treated with engineered T cells that are produced by the methods described herein include, but are not limited to, cancer, viral infection, acute or chronic inflammation, autoimmune disease or any other immune-dysfunction.
  • a “cancer” may be any cancer that is associated with a surface antigen or cancer marker, including, but not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical, carcinoma, AIDS-related cancers, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumors, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumors, central nervous system cancers, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelogenous leuk
  • the cancer is a B cell malignancy.
  • B cell malignancies include, but are not limited to, Non-Hodgkin's Lymphomas (NHL), Diffuse Large B Cell Lymphoma (DLBCL), Small lymphocytic lymphoma (SLL/CLL), Mantle cell lymphoma (MCL), Follicular lymphoma (FL), Marginal zone lymphoma (MZL), Extranodal (MALT lymphoma), Nodal (Monocytoid B-cell lymphoma), Splenic, Diffuse large cell lymphoma, B cell chronic lymphocytic leukemia/lymphoma, Burkitt's lymphoma and Lymphoblastic lymphoma.
  • a “viral infection” may be an infection caused by any virus which causes a disease or pathological condition in the host.
  • viral infections that may be treated with the engineered T cells that are produced by the methods described herein include, but are not limited to, a viral infection caused by an Epstein-Barr virus (EBV); a viral infection caused by a hepatitis A virus, a hepatitis B virus or a hepatitis C virus; a viral infection caused by a herpes simplex type 1 virus, a herpes simplex type 2 virus, or a herpes simplex type 8 virus, a viral infection caused by a cytomegalovirus (CMV), a viral infection caused by a human immunodeficiency virus (HIV), a viral infection caused by an influenza virus, a viral infection caused by a measles or mumps virus, a viral infection caused by a human papillomavirus (HPV), a viral infection caused by a parainflu
  • EBV Epstein
  • a viral infection may lead to or result in the development of cancer in a subject with the viral infection (e.g., HPV infection may cause or be associated with the development of several cancers, including cervical, vulvar, vaginal, penile, anal, oropharyngeal cancers, and HIV infection may cause the development of Kaposi's sarcoma).
  • HPV infection may cause or be associated with the development of several cancers, including cervical, vulvar, vaginal, penile, anal, oropharyngeal cancers, and HIV infection may cause the development of Kaposi's sarcoma.
  • chronic inflammation diseases, autoimmune diseases or any other immune-dysfunctions that may be treated with the engineered T cells produced by the methods described herein include, but are not limited to, multiple sclerosis, lupus, and psoriasis.
  • Chronic inflammation diseases include rheumatoid arthritis, allergies, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and Celiac disease.
  • treat may refer to preventing a condition or disease, slowing the onset or rate of development of the condition or disease, reducing the risk of developing the condition or disease, preventing or delaying the development of symptoms associated with the condition or disease, reducing or ending symptoms associated with the condition or disease, generating a complete or partial regression of the condition or disease, or some combination thereof.
  • a “therapeutically effective amount” or a “therapeutically effective dose” is an amount of engineered lymphocytes that produce a desired therapeutic effect in a subject, such as preventing or treating a target condition or alleviating symptoms associated with the condition by killing target cells.
  • the most effective results in terms of efficacy of treatment in a given subject will vary depending upon a variety of factors, including but not limited to the characteristics of the engineered lymphocytes (including longevity, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of any pharmaceutically acceptable carrier or carriers in any composition used, and the route of administration.
  • a therapeutically effective dose of engineered lymphocytes also depends on the cell surface receptor that is expressed by the lymphocytes (e.g., the affinity and density of the cell surface receptors expressed on the cell), the type of target cell, the nature of the disease or pathological condition being treated, or a combination of both.
  • the engineered lymphocytes prepared by the instant process have greatly increased in vivo efficacy and thus a much lower dose is required, as compared to the conventional technology.
  • a therapeutically effective dose of engineered lymphocytes is fewer than about 2 million engineered lymphocytes per kilogram of body weight of the subject in need of treatment (cells/kg). Therefore, in some aspects, a therapeutically effective dose of engineered lymphocytes is from about 10,000 to about 2,500,000 engineered lymphocytes/kg. In certain embodiments, a therapeutically effective dose of engineered lymphocytes is from about to about 1,500,000 engineered lymphocytes/kg. In certain embodiments, the therapeutically effective dose is about 20,000 to about 1,200,000 engineered lymphocytes/kg. In certain embodiments, the therapeutically effective dose is about 20,000 to about 1,000,000 engineered lymphocytes/kg. In certain embodiments, the therapeutically effective dose is about to about 500,000 engineered lymphocytes/kg.
  • the therapeutically effective dose is about 20,000 to about 400,000 engineered lymphocytes/kg. In certain embodiments, the therapeutically effective dose is about 40,000 to about 400,000 engineered lymphocytes/kg. In certain embodiments, the therapeutically effective dose is about 50,000 to about 200,000 engineered lymphocytes/kg. In certain embodiments, the therapeutically effective dose is about 50,000 to about 100,000 engineered lymphocytes/kg.
  • a therapeutically effective dose of engineered lymphocytes is from about 1,600,000 to about 2,500,000 engineered lymphocytes per kilogram of body weight of the subject in need of treatment (cells/kg). In some embodiments, the therapeutically effective dose of engineered lymphocytes is from about 2,000,000 to about 2,400,000 engineered lymphocytes per kilogram of body weight of the subject in need of treatment (cells/kg).
  • the T cells administered are Yescarta® (axicabtagene ciloleucel). In some embodiments, the T cells administered are Tecartus® (brexucabtagene autoleucel).
  • This example describes a process of preparing lymphocytes that are transduced with a polynucleotide vector, such a viral vector, that encodes a therapeutic protein.
  • the prepared lymphocytes can be useful for treating various diseases such as cancer, especially when the therapeutic protein is a chimeric antigen receptor (CAR) or T cell receptor (TCR) designed to target a cancer cell.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • 7-day process and “7-day lymphocyte manufacturing process” are used interchangeably and refer to a CAR cell manufacturing process which takes about 7 days following initial enrichment and activation steps.
  • the 7-day process is at least 8 days in length from the initial enrichment and activation steps to a harvesting step, and can be between 8 to 11 days in total when including the enrichment and activation steps.
  • apheresis Collection White blood cells were collected (leukapheresis) using standard apheresis equipment, such as Cobe® Spectra, Spectra Optia®, FenwalTM Amicus® or equivalent.
  • the leukapheresis process typically yielded approximately 200-400 mL of an apheresis product from patients.
  • the apheresis product may be subjected to the manufacturing process on-site, or optionally shipped at 1-10° C. to a facility to undergo the manufacturing process in a different location. Further process steps may be conducted in an ISO 7 cell culture process suite (or similar clean room type environment).
  • volume reduction step was performed using a cell processing instrument such as the Sepax® 2 laboratory instrument (Biosafe SA; Houston, TX) or equivalent, and carried out using a standard aseptic tubing kit.
  • the volume reduction step is designed to standardize the volume of cells to approximately 120 mL.
  • the volume reduction step need not be performed, and the cells directly carried to the lymphocyte enrichment step.
  • the volume reduction step is designed to standardize the volume of cells received from each subject, retain mononuclear cells, achieve consistent cell yield and high cell viability, and maintain a closed system to minimize risk of contamination.
  • Lymphocyte Enrichment Following the Volume Reduction step, the cells were subjected to Ficoll based separation on a cell processing instrument, such as the Sepax® 2 or equivalent, using the separation protocol developed and recommended by the instrument manufacturer (NeatCell Program) and using a standard aseptic tubing kit.
  • the lymphocyte enrichment step reduces product related impurities such as RBCs, and granulocytes, enriches and concentrates the mononuclear cells, washes and reduces process related residuals such as Ficoll, and formulates the cells in growth media in preparation for cell activation, as well as achieving consistent cell yield and high cell viability.
  • the closed system minimizes environmental contamination.
  • the process may be carried out in an ISO 7 area at ambient temperature and all connections may be conducted either using a sterile tubing welder, or carried out in an ISO 5 laminar flow hood.
  • the Lymphocyte Activation step may be carried out either with freshly processed cells from the Lymphocyte Enrichment, or previously cryopreserved cells. In the event that cryopreserved cells are used, the cells may be thawed using developed protocols prior to use.
  • the Lymphocyte Activation step selectively activates lymphocytes to become receptive to retroviral vector transduction, reduces the viable population of all other cell types, achieves consistent cell yield and high lymphocyte viability, and maintains a closed system to minimize the risk of contamination. Lymphocyte Activation can be achieved with lymphocyte stimulating agents such as anti-CD3 antibodies and IL-2.
  • the Wash 1 step reduces process related residuals such as anti-CD3 antibody, spent growth media, and cellular debris; achieves consistent cell yield and high T cell viability, maintains a closed system to minimize the risk of contamination; and concentrates and delivers a sufficient number of viable T cells in a small volume appropriate for initiation of transduction.
  • Activated cells from the Wash 1 step in of fresh cell growth media were transferred to a cell culture bag (Origen Biomedical PL240 or comparable) which has been previously prepared by first coating the bags with a recombinant fibronectin or fragments thereof such as RetroNectin® (Takara Bio, Japan), and subsequently incubated with retroviral vector according to defined procedures prior to introduction of the activated cells.
  • RetroNectin® coating (10 ⁇ g/mL) was carried out at a temperature of 2-8° C. for 20 ⁇ 4 hr, washed with dilute buffer, and subsequently incubated with thawed retroviral vector for approximately 180-210 min at 37 ⁇ 1° C. and 5 ⁇ 0.5% CO 2 .
  • the retroviral transduction step cultures the activated T cells in the presence of the retroviral vector under controlled conditions in order to allow for efficient transduction to take place, achieves consistent cell yield and high cell viability, and maintains a closed system in order to minimize the risk of contamination.
  • the Wash 2 step is designed to reduce process related residuals such as retroviral vector particles, vector production process residuals, spent growth media, and cellular debris achieve consistent cell yield and high cell viability; maintain a closed system to minimize the risk of contamination; and exchange spent growth media for fresh media with a target number of cells in a specified volume appropriate for initiation of expansion step.
  • Lymphocyte Expansion Cells from the Wash 2 step were aseptically transferred to a culture bag (Origen Biomedical PL325 or equivalent) and diluted with fresh cell growth media and cultured for approximately 72 hr at 37 ⁇ 1° C. and 5 ⁇ 0.5% CO 2 . The cell density was measured daily starting on Day 5. Because doubling times of the T cells may vary slightly from subject to subject, additional growth time beyond 72 hr (i.e., 3-6 days) may be necessary in the event that the total cell number is insufficient to deliver a target dose of CAR-positive T cells/kg of subject weight.
  • the lymphocyte expansion step is designed to culture the cells under controlled conditions in order to produce a sufficient number of transduced cells for delivering an efficacious dose, maintain a closed system to minimize risk of contamination, and achieve consistent cell yield and high cell viability.
  • One such efficacious dose or target dose includes 2 ⁇ 10 6 FMC63-28Z CAR positive or FMC63-CD828BBZ CAR positive T cells/kg ( ⁇ 20%) of subject weight that were produced via transduction with either the MSGV-FMC63-28Z retroviral vector or the MSGV-FMC63-CD828BBZ retroviral vector, respectively, both of which are described in detail in Kochenderfer et al., J Immunother. 2009 September; 32(7): 689-702.
  • wash 3 and Concentrate Following the lymphocyte expansion step, the cells were washed with 0.9% saline using a cell processing instrument, such as the Sepax® 2 or equivalent, in a standard aseptic kit using developed protocols by the manufacturer, and the cells were concentrated to a final volume of approximately 35 mL in preparation for the formulation and cryopreservation.
  • the wash 3 step is designed to reduce process related residuals such as retroviral production process residuals, spent growth media, and cellular debris; achieve consistent cell yield and high cell viability; and maintain a closed system to minimize risk of contamination.
  • an appropriate cell dose may be formulated for preparation of the final cryopreserved product.
  • the embodiments described herein provide for efficient production of an engineered lymphocyte therapy within 7 days.
  • This example developed an expedited process based on the 7-day process as described in Example 1.
  • 5-day process and “5-day lymphocyte manufacturing process” are used interchangeably and refer to a CAR cell manufacturing process which takes about 5 days following initial enrichment and activation steps.
  • the 5-day process is 6 days in length from the initial enrichment and activation steps to a harvesting step, and can be between 6 to 9 days in total when including the enrichment and activation steps.
  • lymphocytes are enriched and activated on day 0; transduction bag is coated with recombinant fibronectin on day 1; viral transduction is conducted on day 2; transduced lymphocytes are washed and then expanded on days 3 and 4; expansion continues with media changed each day on day 5 and 6; and the final cell products are harvested on day 7. From about 1.2 ⁇ 10 9 lymphocytes acquired from apheresis, about 2.4 ⁇ 10 8 lymphocytes are incubated with the viral vectors for transduction.
  • the Origen Biomedical PL240 bag was used for transduction, or more preferably, a PL750 bag.
  • the larger bag allowed a larger volume of the vector (200 mL instead of 100 mL) and more lymphocytes (between 3.2 ⁇ 10 8 and 6 ⁇ 10 8 instead of 2.4 ⁇ 10 8 ) used in the transduction step.
  • the in vivo efficacy of the cell products from the 5-day process was tested in a mouse model implanted with 5 ⁇ 10 5 Nalm6 cells (a human acute lymphoblastic leukemia (ALL) line) in a 100 ⁇ L solution, iv.
  • the CAR used, injected on day 6, targeted CD19, at doses of 5 ⁇ 10 6 cells, 1 ⁇ 10 6 cells, or 0.2 ⁇ 10 6 cells/kg body weight (Table 3).
  • the tumor inhibition results are shown in Table 5 (measured as photons/s).
  • the cell products from the 5-day process When dosed at 5 ⁇ 10 6 cells/kg, the cell products from the 5-day process showed complete tumor growth inhibition. At all doses, the cells products from the 5-day process outperformed those from the 7-day process. Cells from the 5-day process were also more efficacious than those of the 7-day process, even when the percentage of CAR+ cells was matched (i.e., fewer CAR-T cells from the 5-day process).
  • This example developed a further expedited process based on the 7-day process as described in Example 1 and the 5-day process as described in Example 2.
  • 3-day process and “3-day lymphocyte manufacturing process” are used interchangeably and refer to a CAR cell manufacturing process which takes about 3 days from initial enrichment and activation steps.
  • the 3-day process is about 4 days in length from the initial enrichment and activation step to a harvesting step.
  • the 3-day process does not include a cell expansion step comprising one or more days following a transduction step and preceding a harvesting step.
  • the day 0-1 procedures are similar to the 5-day process, including the fibronectin-coating of a larger bag (e.g., Origen Biomedical PL325 or preferably PL750) for the subsequent transduction on day 2.
  • a larger bag e.g., Origen Biomedical PL325 or preferably PL750
  • lymphocytes were used in the transduction step, with the same amount of viral vectors (200 mL).
  • Another important difference is that, unlike in the 5-day and 7-day processes, no specific step of T cell expansion was conducted. Instead, the transduced lymphocytes were harvested on day 3, allowing the entire process to be completed within 3 days from the initial enrichment and activation steps.
  • the cell products harvested included a slightly smaller percentage of T cells (CD3+).
  • the 3-day products included an even greater percentage of juvenile (na ⁇ ve) T cells (Table 6). Note that in Table 6 below, historical averages from 7-day process runs are reflected in the column labeled “Historical averages”, and data from the 3-day and 7-day runs conducted in the current study are depicted in the columns labeled “3-day” and “7-day,” respectively.
  • the 3-day process generates roughly a 1.4-fold increase in the percentage of CD4+ na ⁇ ve T cells versus the percentage of such cells observed from the 5-day process, and roughly an 9.5-fold increase in the percentage of CD8+ na ⁇ ve T cells versus the percentage of such cells observed from the 5-day process. Also, the 3-day process generates roughly a 3.0-fold increase in the percentage of CD4+ na ⁇ ve T cells versus the historical average of such cells observed from the 7-day process, and roughly an 18.0-fold increase in the percentage of CD8+ na ⁇ ve T cells versus the historical average of such cells observed from the 7-day process.
  • the 3-day process generated only about 4.35% effector memory T cells while the 5-day process generated about 8.55%; within CD8+ T cells, the 3-day process only generated about 9.85% effector memory T cells while the 5-day process generated about 15.85%.
  • the 3-day process generates roughly a 2.0-fold decrease in the percentage of CD4+ effector memory T cells versus the percentage of such cells observed from the 5-day process, and roughly an 1.6-fold decrease in the percentage of CD8+ effector memory T cells versus the percentage of such cells observed from the 5-day process. Also, the 3-day process generates roughly a 6.0-fold decrease in the percentage of CD4+ effector memory T cells versus the historical average of such cells observed from the 7-day process, and roughly a 3.5-fold decrease in the percentage of CD8+ effector memory T cells versus the historical average of such cells observed from the 7-day process.
  • the percentage of CD4+ CCR7+ cells (i.e. CM and Naive cells) in the final harvested population from the 3-day process is at least 95% of the harvested CD4+ T-cell population.
  • the maximum percentage of Teff/TEMRA and EM CD4+ cells in the final harvested population from the 3-day process is about 5%.
  • the percentage of CD4+ CCR7+ cells (i.e. CM and Naive cells) in the final harvested population from the 7-day process is about 71% of the harvested CD4+ T-cell population.
  • the maximum percentage of Teff/TEMRA and EM CD4+ cells in the final harvested population from the 7-day process is about 29%.
  • the 3-day process generates a population of CD4+ T-cells with about a 1.3-fold increase in the percentage of CD4+ CCR7+ cells as the 7-day process, and with about a 5-fold decrease in the percentage of Teff/TEMRA and EM CD4+ cells.
  • the percentage of CD8+ CCR7+ cells (i.e. CM and Naive cells) in the final harvested population from the 3-day process is at least 84% of the harvested CD8+ T-cell population.
  • the maximum percentage of Teff/TEMRA and EM CD4+ cells in the final harvested population from the 3-day process is about 16%.
  • the percentage of CD8+ CCR7+ cells (i.e. CM and Naive cells) in the final harvested population from the 7-day process is about 66% of the harvested CD8+ T-cell population.
  • the maximum percentage of Teff/TEMRA and EM CD8+ cells in the final harvested population from the 7-day process is about 34%.
  • the 3-day process generates a population of CD8+ T-cells with about a 1.3-fold increase in the percentage of CD8+ CCR7+ cells as the 7-day process, and with about a 2-fold decrease in the percentage of Teff/TEMRA and EM CD8+ cells.
  • This example compared the in vivo efficacy of cells prepared by the 3-day process as described in Example 3, to those from the 7-day process, with the same animal model and procedure as described in Example 2.
  • the tumor inhibition results are shown in Table 10 (measured as photons/s).
  • CAR Both types of CAR included a CD19-binding antigen binding fragment, while CAR #1 included a CD28 co-stimulatory domain, and CAR #2 included a 4-1BB co-stimulatory domain, both delivered by a lentiviral vector.
  • Cells were harvested at day 4, day 7 and day 14, and doses for the in vivo animal study included 0.2 ⁇ 10 6 cells/kg and 1 ⁇ 10 6 cells/kg (Table 12). The results are shown in Table 13.
  • a CAR that targets CD19 and includes a CD28 costimulatory domain was tested in vivo with the Nalm6 mouse model of leukemia.
  • the results showed that early harvesting (day 4) led to better in vivo anti-tumor efficacy than regular harvesting (day 7), which in turn was more efficacious than late harvesting (day 14).
  • a low dose (2 ⁇ 10 5 cells) of T cells harvested on day 4 was 5 times more efficacious than a high dose (10 6 cells) of T cells harvested on day 7.
  • Example 4 complements Example 4 and presents additional data comparing the in vivo efficacy of anti-CD19 CART cells prepared by the 3-day process described in Example 3, to those prepared by the 7-day process.
  • Anti-CD19 CAR T cells prepared by the 3-day process and anti-CD19 CAR T cells prepared by the 7-day process along with their respective non-transduced (NTD) Day 3 and NTD Day 7 cells were manufactured from a healthy human donor.
  • NALM6-luc cells (5 ⁇ 10 5 cells) were implanted IV via the lateral tail vein into 8-week-old female NSG mice (5 mice per group).
  • mice received either controls (NTD Day 3 or NTD Day 7) in a single dose cohort or anti-CD19 CAR T-cell products as multiple dose cohorts.
  • Antitumor activity was assessed by determining the effects of anti-CD19 CAR T-cell products on tumor burden and animal survival.
  • Antitumor activity was evaluated by assessing the change in tumor burden as measured by bioluminescence imaging (BLI) from Day 5 (1 day before CAR T-cell treatment) to Day 22 (16 days after CAR T-cell treatment, and the last time point in which all groups were intact). Inhibition of tumor growth was observed in all 4 dose cohorts of mice treated with anti-CD19 CAR T cells derived from the 7-day process and all 5 dose cohorts of anti-CD19 CAR T cells derived from the 3-day process (Tables 14, 15, and 16).
  • tumor growth inhibition compared with the no treatment group was 128.1%, 126.7%, 117.7%, 113.4%, and 65.7% in animals treated with 1 ⁇ 10 6 , 5 ⁇ 10 5 , 3 ⁇ 10 5 , 2 ⁇ 10 5 , and 1 ⁇ 10 5 CAR + T cells, respectively.
  • tumor growth inhibition ranged between 109.0% to 115.1% for the dose cohorts tested (Table 16).
  • tumor burden (measured by BLI) was login normalized, with means for each cohort at Day 5 and Day 20 of the study represented in the table.
  • the change in tumor BLI from Day 5 to Day 20 was calculated, and the percent change in tumor burden relative to the no treatment group is calculated as ⁇ T/ ⁇ C.
  • Tumor growth inhibition was defined as (1-[ ⁇ T/ ⁇ C] ⁇ 100) and represented the percentage tumor volume change in treatment arms relative to the control (ie, mice that received no treatment).
  • a Tumor Growth Inhibition (TGI) of 0% indicates that mean tumor growth of the group was comparable to the mean tumor growth observed in the no treatment group, while 100% TGI indicates no tumor growth was observed between Day 5 to Day 20.
  • a TGI >100% indicates that the mean tumor burden regressed from Day 5 to Day 20, while a TGI ⁇ 0 indicates a tumor burden increase greater than the increase seen in the no treatment group (G1).
  • mice with anti-CD19 CAR T cells prepared by the 3-day process or anti-CD19 CAR T cells prepared by the 7-day process resulted in prolonged survival compared with control mice that received NTD or no treatment.
  • groups treated with anti-CD19 CAR T cells prepared by the 3-day process cells experienced significantly greater survival, with the mice from all dose cohorts achieving 100% survival at the study end point on Day 64.
  • mice treated with anti-CD19 CART cells prepared by the 7-day process had lower survival in comparison to groups treated with anti-CD19 CAR T cells prepared by the 3-day process, ranging from 80% survival for the 1 ⁇ 10 7 CAR + T-cell dose cohort to 20% survival for the 5 ⁇ 10 6 CAR′ T-cell dose cohort and 0% survival for the 3 ⁇ 10 6 and 1 ⁇ 10 6 CAR′ T-cell dose cohorts.
  • mice that received anti-CD19 CAR T cells prepared by the 3-day process at all doses achieved a statistically significant improvement in response relative to those which received the anti-CD19 CAR T cells prepared by the 7-day process at the 3 ⁇ 10 6 and 1 ⁇ 10 6 CAR′ T-cell doses, but were not statistically different from the 1 ⁇ 10 7 CAR′ T-cell dose of anti-CD19 CAR T cells prepared by the 7-day process.
  • This example describes functional and phenotypic characterization of anti-CD19/CD20 CAR T-cells that were harvested on day 3 as compared to anti-CD19/CD20 CAR T-cells that were harvested on day 6.
  • CD4+ and CD8+ T cells from a healthy human donor were activated and transduced using a lentiviral vector encoding a dual targeting anti-CD19 CAR and anti-CD20 CAR.
  • the transduced cells were subsequently harvested on Day 3.
  • another set of anti-CD19/CD20 CAR T-cell products were manufactured from T cells from the same donor but harvested on Day 6 (referred to in Examples 7 and 8 as “anti-CD19/CD20 CAR Day 6”).
  • both CAR T-cell products were functionally characterized and compared with their respective NTD control T cells (NTD Day 3 and NTD Day 6) that were manufactured in parallel from the same donor material.
  • the anti-CD19/CD20 CAR T-cells that were harvested on day 3 were produced by the following 3-day process beginning with apheresis material collection.
  • the starting apheresis material may optionally be fresh or cryopreserved apheresis or cryopreserved T cells.
  • Wash 1, wash 2, T-cell enrichment, and wash 3 steps were conducted on day 0.
  • T cell activation was conducted on day 0 up to day 1.
  • Lentiviral transduction was conducted on day 1 up to day 4.
  • Harvest wash and concentrate was conducted on day 3 up to day 4.
  • the anti-CD19/CD20 CAR T-cells that were harvested on day 6 were produced by the following process beginning with apheresis material collection. Wash 1, wash 2, T-cell enrichment, and wash 3 steps were conducted on day 0. T cell activation was conducted on day 0 up to day 2. Lentiviral transduction was conducted on day 2 up to day 5. Wash 4 was conducted on day 4 up to day 5. T-cell expansion was conducted on day 4 up to day 15. Harvest wash and concentrate was conducted on day 6 up to day 15.
  • the anti-CD19/CD20 CAR T-cells produced by the 3-day process and the anti-CD19/CD20 CAR Day 6 T-cell products were assessed for CAR cell-surface expression to establish CAR transduction efficiencies.
  • Cell-surface expression of both the anti-CD19 CAR and anti-CD20 CAR were individually detected by flow cytometry using fluorescent-labelled anti-idiotypic antibodies.
  • Individual expression of the anti-CD19 CAR was 68% for anti-CD19/CD20 CAR T-cells produced by the 3-day process and 35% for the anti-CD19/CD20 CAR Day 6 T-cell product, and expression of the antiCD20 CAR was 65% and 37%, respectively, in the same samples.
  • Transduction efficiency as measured by the sum of total anti-CD19 and antiCD20 CAR expression (i.e., percentage of anti-CD19 CAR antibody+ cells plus percentage of anti-CD29 CAR antibody cells), was 72% for anti-CD19/CD20 CAR T-cells produced by the 3-day process and 40% for the antiCD19/CD20 CAR Day 6 T-cell product.
  • CD45RA and CCR7 markers may be used to define the various populations of T cells, including na ⁇ ve and stem memory T cells (CD45RA + CCR7 + ), also referred to as juvenile T cells.
  • the anti-CD19/CD20 CAR T-cells produced by the 3-day process showed a slightly higher CD4/CD8 ratio than the anti-CD19/CD20 CAR Day 6 T-cell product (Table 18).
  • CD4/CD8 ratio (% of CD3) CD4 CD8 G1 55.8 41.4 G2 53.8 42.1 G3 51.9 47.1 G4 49.2 48.6
  • Both CD4+ and CD8+ populations of anti-CD19/CD20 CAR T-cells produced by the 3-day process showed an increased frequency of juvenile cells compared with the anti-CD19/CD20 CAR Day 6 T-cell product (Table 19 and Table 20).
  • CAR T-cell products were assessed in co-culture assays with antigen-positive and antigen-negative target cells.
  • NTD T-cell products were included in the experiments as controls to assess levels of alloreactivity. Cytotoxicity was measured at 1 day and 4 days after initiation of co-culture of the T-cell products with luciferase-expressing target cells. Cytotoxicity was measured as the reduction of the luciferase signal emitted in the test wells upon addition of luciferin compared with the wells where target cells alone were seeded. Multiple E:T ratios were used in these assays ranging from 1:1 to 1:243.
  • Raji a B-cell lymphoma cell line that expresses high levels of CD19 and CD20 antigens
  • Nalm6 a B-cell leukemia cell line that expresses high levels of CD19 but low levels of CD20
  • ST486 a B-cell lymphoma cell line that expresses low levels of CD19 but high levels of CD20
  • K-562 a chronic myelogenous leukemia cell line that does not express CD19 or CD20 antigens.
  • controls consisted of Raji CD19 knockout (KO) cells, and Raji CD20KO cells.
  • Raji cell lines were engineered to express either antigen: Raji CD19KO cells only express CD20 and not CD19, Raji CD20KO only express CD19 and not CD20.
  • Antigen KO was confirmed at the DNA level by tracking of insertions or deletions (indels) by decomposition (TIDE) analysis and at the cell-surface protein level by flow cytometry.
  • Specific activity was further evaluated by measurement of cytokine levels in culture supernatants after an overnight co-culture of T-cell products with Nalm6, ST486, Raji, Raji CD19KO, Raji CD20KO, and K-562 target cells at an E:T ratio of 1:1. Quantitation of IFN- ⁇ , IL-2, tumor necrosis factor- ⁇ (TNF- ⁇ ) pro-inflammatory cytokine production was performed.
  • anti-CD19/CD20 CAR T-cells produced by a three-day process and anti-CD19/CD20 CAR Day 6 T-cell products showed robust antigen-dependent IFN- ⁇ , IL-2, and TNF- ⁇ pro-inflammatory cytokine production from co-culture with the antigen-positive target cells, Nalm6, ST486, Raji, Raji CD19KO, and Raji CD20KO (Table 22A-22C).
  • Both anti-CD19/CD20 CAR T-cells produced by a three-day process and anti-CD19/CD20 CAR Day 6 T-cell products showed comparable cytokine levels of IL-2 and TNF- ⁇ , across the various target cell lines tested.
  • each individual CAR was demonstrated by the production of cytokines when the T-cell products were co-cultured with target cells expressing a single antigen (Raji CD19KO or Raji CD20KO), albeit at lower levels than that induced by co-culture with Raji parental cells, which express both antigens.
  • target cells i.e., T cells alone
  • antigen-negative K-562 CD19 ⁇ CD20 ⁇
  • T-cell products that had been co-cultured with Raji, Nalm6, Raji CD19KO, Raji CD20KO, and K-562 target cells were harvested 4 days after initiation of co-culture, centrifuged to pellets, and prepared for flow cytometry evaluation.
  • T cells had been prelabeled with a fluorescent dye used to trace multiple generations of cells by analysis of dye dilution upon cell division. The 1:1 E:T ratio from the co-cultures was used and the T-cell products that had been cultured in the absence of target cells (T cells alone) were used as controls.
  • T cells co-cultured with antigen-negative K-562 (CD19 ⁇ CD20 ⁇ ) cells were used to assess specificity and levels of antigen-independent proliferation while T cells alone were used to assess basal levels of proliferation in the absence of stimuli.
  • cells were stained with fluorescent-labeled antibodies against CD3, CD4, CD8, CD19 CAR, CD20 CAR, CD25 and a viability dye.
  • Anti-CD19/CD20 CAR T-cells produced by a three-day process and anti-CD19/CD20 CAR Day 6 T-cell products showed comparable proliferation in co-culture with Nalm6, Raji, Raji CD19KO, and Raji CD20KO target cells, but not with antigen-negative K-562 cells, demonstrating that both the anti-CD19 CAR and anti-CD20 CAR in the T-cell products are functional.
  • the NTD T-cell controls showed various levels of nonspecific basal proliferation when co-cultured with the different target cell lines, but overall, the NTD Day 3 cells demonstrated more basal proliferation than the NTD Day 6 cells.
  • Nalm6luc cells (5.0 ⁇ 10 5 ) were implanted intravenously via the lateral tail vein into 7 week-old female NSG mice (5 mice per group). On Day 6 after tumor implantation, mice received either controls (vehicle [phosphate-buffered saline] or NTD Day 3 cells) or anti-CD19/CD20 CAR T cells, as follows: Anti-CD19/CD20 CAR T-cells produced by a three-day process at 1 of 3 dose levels (2.0 ⁇ 10 5 , 4.0 ⁇ 10 4 , and 8.0 ⁇ 10 3 CAR + T cells); antiCD19/CD20 CAR Day 6 T-cell product at 1 dose level (2.0 ⁇ 10 5 CAR + T cells). All animals were dosed with a single IV administration of applicable treatment at a fixed volume of 100 ⁇ L and mice from the vehicle group received an IV injection of 100 ⁇ L PBS. Table 23 shows the experimental groups for the results represented in Tables 24-26.
  • Antitumor activity was evaluated by assessing the change in tumor burden (measured by bioluminescence and log 10 normalized) from Day 5 (1 day before CAR T-cell infusion) to Day 22 (i.e., 16 days after CAR T-cell infusion, and the last time point at which all groups were intact).
  • Treatment with anti-CD19/CD20 CAR T-cells produced by a three-day process resulted in a significant reduction in tumor burden when compared with mice that received either vehicle (PBS) or NTD Day 3 T cells (Table 24).
  • Tumor burden (measured by BLI) was login normalized, with means for each cohort at Day 5 and Day 22 of the study represented in Table 25.
  • the change in tumor BLI from Day 5 to Day 22 was calculated, and the percentage change in tumor burden relative to the vehicle group was calculated as ⁇ T/ ⁇ C.
  • Tumor growth inhibition was defined as (1-[ ⁇ T/ ⁇ C] ⁇ 100) and represented the percentage tumor volume change in study arms relative to the control (ie, mice that received vehicle [PBS]).
  • a TGI of 0% indicates that the mean tumor growth of the group was comparable to the mean tumor growth observed in mice that received vehicle, while 100% TGI indicates no tumor growth was observed between Day 5 to Day 22.
  • a TGI >100% indicates that the mean tumor burden regressed from Day 5 to Day 22, while a TGI ⁇ 0 indicates a tumor burden increase greater than the increase seen in mice that received vehicle.
  • Antitumor efficacy as determined by the analysis of tumor burden, reached statistical significance on Day 22 for the mice treated with anti-CD19/CD20 CAR T-cells produced by a three-day process at all doses tested (2.0 ⁇ 10 5 , 4.0 ⁇ 10 4 , and 8.0 ⁇ 10 3 CAR + T cells) and for the mice treated with the anti-CD19/CD20 CAR Day 6 product at 2.0 ⁇ 10 5 CAR + T cells (Table 26).
  • mice with anti-CD19/CD20 CAR T-cells produced by a three-day process or anti-CD19/CD20 CAR Day 6 T-cell products resulted in increased overall survival (OS) compared with mice that received vehicle or NTD controls. All mice that received either vehicle or NTD controls exited the study by Day 15 or Day 12, respectively, based on the survival setpoint defined by tumor burden ⁇ 1 ⁇ 10 10 photons/second bioluminescence. Groups treated with anti-CD19/CD20 CAR T-cells produced by a three-day process at the highest doses of 2.0 ⁇ 10 5 and 4.0 ⁇ 10 4 CAR + T cells achieved 80% and 100% survival, respectively, at the study end point.
  • mice treated with the lowest dose of anti-CD19/CD20 CAR T-cells produced by a three-day process reached a median survival of 12 days, similar to the mice from the control groups (vehicle and NTD Day 3).
  • mice that received anti-CD19/CD20 CAR Day 6 T-cell product had a reduced median survival (36 days) when compared with mice treated with anti-CD19/CD20 CAR T-cells produced by a three-day process (median survival point not achieved).
  • treatment with anti-CD19/CD20 CAR T-cells produced by a three-day process significantly delayed tumor growth and increased survival in a Nalm6-luc human B-ALL xenograft NSG mouse model.
  • This example complements Example 3 and presents additional data related to the phenotypic characteristics of the harvested cells manufactured using the 3-day process.
  • Tables 27 and 28 show final product data for cells prepared using the 3-day process.
  • the cells were derived from healthy donor material (fresh apheresis or frozen PBMCs; N1-N9) and from patient material (frozen PBMCs; P1-P4).
  • the final product runs exhibited higher frequencies of central memory (CM) and juvenile cells compared to Teff/TEMRA and effector memory (EM) cell populations, indicating a more juvenile product (Tables 27 and 28).
  • CM central memory
  • EM effector memory
  • HD healthy donor
  • TEMRA terminally differentiated effector memory T cells.
  • CD45RA and CCR7 were used to define T cell phenotype subsets, which include juvenile T cells (Na ⁇ ve, CD45RA+CCR7+), CM (CD45RA ⁇ CCR7+), EM (CD45RA ⁇ CCR7 ⁇ ), and Teff/TEMRA (CD45RA+CCR7 ⁇ )
  • the percentage of CD4+ CCR7+ cells (i.e. CM and Juvenile cells) in the final harvested product is at least 80% of the harvested CD4+ T-cell population. Put another way, the maximum percentage of Teff/TEMRA and EM CD4+ cells in the final harvested population is about 20%. Thus, there are at least 4 times as many CD4+ CCR7+ cells in the harvested population as Teff/TEMRA and EM CD4+ cells.
  • the percentage of CD8+ CCR7+ cells (i.e. CM and Juvenile cells) in the final harvested product is at least about 60% of the harvested CD8+ T-cell population. Put another way, the maximum percentage of Teff/TEMRA and EM CD8+ cells in the final harvested population is about 40%. Thus, there are at least 1.5 times as many CD8+ CCR7+ cells in the harvested population as Teff/TEMRA and EM CD8+ cells.
  • This example describes functional and phenotypic characterization of anti-CLL-1 CAR T-cells that were harvested on day 3 as compared to anti-CLL-1 CAR T-cells that were harvested on day 8.
  • the anti-CLL-1 CAR T cells described in this example were produced by a similar method as described in Example 7.
  • T cells selected from a healthy human donor were activated on day 0 and transduced on day 1 using a lentiviral vector encoding an anti-CLL-1 CAR.
  • the transduced cells were subsequently harvested on Day 3.
  • Another set of anti-CLL-1 CAR T-cells were manufactured from T cells from the same donor but harvested on Day 8. Subsequently, both CAR T-cell products were functionally characterized and compared with their respective NTD control T cells (NTD Day 3 and NTD Day 8).
  • the anti-CLL-1 CAR T cells harvested on day 3 and day 8 were assessed for CAR cell-surface expression to establish CAR transduction efficiencies. CAR expression was comparable between those harvested on day 3 and day 8. Expression of the anti-CLL-1 CAR was 60.4% for the anti-CLL-1 CAR T-cells harvested on day 3 and 78.9% for the anti-CLL-1 CAR T-cells harvested on day 8.
  • Phenotypic characterization of CAR T-cell products was performed by flow cytometry.
  • the anti-CLL-1 CAR T cells harvested on day 3 showed an increased juvenile T cell profile with 83.7% CD45RA + CCR7 + cells on day 3 harvest compared to 23% CD45RA + CCR7 + on day 8 harvest.
  • Anti-CLL-1 CAR T cells harvested on day 3 displayed increased CD4+ cells compared to those harvested on day 8 as shown in Table 30.
  • CAR T-cell products were assessed in co-culture assays with antigenpositive target cells.
  • NTD T-cell products were included in the experiments as controls. Cytotoxicity was measured at 1 day and 4 days after initiation of coculture of the T-cell products. E:T ratios of 1:1 to 1:3 were used.
  • the MV4-11 (CLL-1 medium expression) and Kasumi-1 (CLL-1 very low expression) cell lines were used.
  • Anti-CLL-1 CAR T-cells harvested on day 3 showed increased cytotoxicity in vitro against Kasumi-1 cells compared to anti-CLL-1 CAR t cells harvested on day 8 (Tables 30A-30B).
  • Anti-CLL-1 CAR T-cells harvested on day 3 and day 8 showed comparable cytotoxicity in vitro against MV4-11 cells (Tables 30C-30D).
  • Phenotypic and functional characteristics of anti-CD19/CD20 CAR T cells produced by a process having a lentivirus transduction step on day 1 or day 2 were compared. In both processes, T cell selection and activation occurred on day 0. CAR expression was measured by detecting an anti-CD19 CAR antibody. Table 35 shows CAR expression of transduced T cells on day 1 and day 2 in cells harvested on day 3 to day 8.
  • T cell subpopulation of CD27+CD28+CD45RA+CCR7+CD62L+ was measured for anti-CD19/CD20 CAR T cells transduced on day 1 and day 2.
  • Table 36 shows the percentage of juvenile CAR+ T cells produced by each method.

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